EP1639091B1 - Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450 - Google Patents

Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450 Download PDF

Info

Publication number
EP1639091B1
EP1639091B1 EP04776528A EP04776528A EP1639091B1 EP 1639091 B1 EP1639091 B1 EP 1639091B1 EP 04776528 A EP04776528 A EP 04776528A EP 04776528 A EP04776528 A EP 04776528A EP 1639091 B1 EP1639091 B1 EP 1639091B1
Authority
EP
European Patent Office
Prior art keywords
mutant
enzyme
hydroxylation
alkane
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP04776528A
Other languages
English (en)
French (fr)
Other versions
EP1639091A4 (de
EP1639091A2 (de
Inventor
Frances H. Arnold
Mathew W. Gevo Incl. PETERS
Peter Meinhold
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CALIFORNIA UNIVERSITY OF TECHNOLOGY
Original Assignee
CALIFORNIA UNIVERSITY OF TECHNOLOGY
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CALIFORNIA UNIVERSITY OF TECHNOLOGY filed Critical CALIFORNIA UNIVERSITY OF TECHNOLOGY
Publication of EP1639091A2 publication Critical patent/EP1639091A2/de
Publication of EP1639091A4 publication Critical patent/EP1639091A4/de
Application granted granted Critical
Publication of EP1639091B1 publication Critical patent/EP1639091B1/de
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)

Definitions

  • the invention relates to variants of cytochrome P450 enzymes that display altered and improved enantio- and regioselectivity in their hydroxylation of alkanes.
  • the invention also relates to novel variants of cytochrome P450 enzymes that are capable of hydroxylating ethanes.
  • Cytochrome P450s are a large superfamily of enzymes that primarily hydroxylate substrates using dioxygen, although other redox-type reactions, including some reductions, have been reported.
  • cytochrome P450 BM-3 is found in the bacterium Bacillus megaterium (EC 1.14.14.1).
  • This variant also known as CYP102, is a water-soluble, catalytically self-sufficient P450 containing a monooxygenase domain (64 kD) and a reductase domain (54 kD) in a single polypeptide chain ( Narhi and Fulco, Journal of Biological Chemistry, 261 (16): 7160-7169 (1986 ) and Journal of Biological Chemistry, 262 (14): 6683-6690 (1987 ); Miura and Fulco, Biochimica et Biophysica ACTA, 388 (3): 305-317 (1975 ); Ruettinger et al., 1989).
  • the minimum requirements for activity of the BM-3 variant are substrate, dioxygen and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH).
  • Nucleotide and amino acid sequences for P450 BM-3 can be found in, the GenBank database under the accession Nos. J04832 ( SEQ ID NO: 1 ) and P14779 ( SEQ ID NO: 2 ), respectively.
  • P450 BM-3 hydroxylates fatty acids of chain lengths between C12 and C18 at subterminal positions, and the regioselectivity of oxygen insertion depends on the chain length ( Miura and Fulco, Biochimica et Biophysica ACTA 388 (3): 305-317 (1975 ); Boddupalli et al., Journal of Biological Chemistry 265 (8): 4233-4239 (1990 )).
  • the natural substrates of P450 BM-3 are hydroxylated at their ⁇ -1, ⁇ -2, and ⁇ -3 positions using atmospheric dioxygen and nicotinamide adenine dinucleotide phosphate (NADPH) as shown in FIG. 1 .
  • Substrate is bound and hydroxylated in a hydrophobic binding pocket that is positioned directly above a heme cofactor which is located in its own domain of the protein.
  • a single peptide chain connects this heme domain to the reductase domain of the protein where NADPH is reduced and flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors are used to transfer electrons to the heme active site for catalysis.
  • FMN flavin mononucleotide
  • FAD flavin adenine dinucleotide
  • the optimal chain length of saturated fatty acid substrates for P450 BM-3 is 14-16 carbons, and the enzyme was initially believed to have no activity towards fatty acids smaller than C12 ( Miura and Fulco, Biochimica et Biophysica ACTA, 388 (3): 305-317 (1975 )).
  • P450 BM-3 is also known to hydroxylate the corresponding fatty acid amides and alcohols and forms epoxides from unsaturated fatty acids ( Miura and Fulco, Biochimica et Biophysica ACTA, 388 (3): 305-317 (1975 ); Capdevila et al., J. Biol. Chem. 271:22663-22671 (1996 ); Graham-Lorence et al., J.
  • Pseudomonas oleovorans is able to oxidize n-alkanes using hydroxylase machinery comprising an integral membrane oxygenase (omega-hydroxylase), a soluble NADH-dependent reductase and a soluble metalloprotein (rubredoxin) which transfers electrons from the reductase to the hydroxylase ( Staijen et al., European Journal of Biochemistry, 267 (7): 1957-1965 (2000 )).
  • the omega-hydroxylase has been cloned from P. oleovorans into E .
  • WO 03/008563 discloses specific nucleic acids encoding cytochrome P450 BM-3 variants reported to have a higher alkane-oxidation capability and higher organic-solvent resistance than wild-type P450 BM-3. Peters M.W., et al.
  • Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant.
  • an isolated mutant P450 enzyme comprising a sequence as set forth in SEQ ID NO: 2 and having the mutations R47C, V78A, A82L, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, and L353V is provided.
  • Said mutant P450 enzyme hydroxylates alkanes.
  • said enzyme is a mutant of the cytochrome P450 BM-3 enzyme.
  • the isolated mutant P450 further comprises a mutation at A328V.
  • the isolated mutant P450 exhibits selective hydroxylation.
  • the isolated mutant P450 comprises selective activity consistent across hexanes, decanes, heptanes, octanes, and nonanes.
  • the isolated mutant P450 may comprise alkane hydroxylation activity that results in the same regiospecific product for hexane, heptane, octane, nonane, and decane for at least 40% of the total products produced from the alkane hydroxylation activity.
  • the isolated mutant P450 may comprise a total turnover for octane of more than about 1000.
  • the isolated mutant P450 may comprise a maximum rate of catalysis of octane of at least about 2001(min -1 ).
  • said mutant BM-3 P450 enzyme has an altered enantiospecificity compared to the enantiospecificity of mutant 139-3 comprising SEQ ID NO:2 having the mutations V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A295T, and L353V; or has an altered regiospecificity compared to the regiospecificity of mutant 139-3.
  • the isolated mutant P450 enzyme may also comprise alkane hydroxylation activity against ethane.
  • a method of making a mutant P450 enzyme with altered selective hydroxylation abilities comprises the steps of providing a first mutant P450 that is capable of alkane hydroxylation of a substrate to produce a product with a first hydroxylation profile; and modifying at least one amino acid in said first mutant P450 to produce a second mutant P450.
  • Said second mutant P450 is capable of alkane hydroxylation of the substrate to produce a product with a second hydroxylation profile, wherein the second mutant comprises the following mutations R47C, V78A, A82L, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, and L353V.
  • the modification of the at least one amino acid is achieved through directed evolution; or the modification of the at least one amino acid is achieved through selected point mutations, preferably, the point mutations are selected from an active site of the mutant P450.
  • an isolated nucleic acid encoding a cytochrome P450 mutant is provided.
  • a method of hydroxylating an alkane in a selective manner comprises the steps of: providing an isolated mutant P450 enzyme of the first aspect of the present invention; and contacting said isolated mutant P450 with said alkane, whereby the isolated mutant P450 hydroxylates the alkane in a selective manner.
  • the selective manner is a regioselective manner; or the selective manner is an enantioselective manner.
  • the isolated mutant P450 hydroxylates said alkane with a turnover number of more than 100.
  • the alkane is an octane and the isolated mutant P450 hydroxylates said octane with a turnover number of more than 1000; or the alkane is selected from the group consisting of decane, nonane, octane, heptane, hexane, pentane, propane, and ethane.
  • FIG. 1 is an illustration of a prior art general hydroxylation reaction of myristic acid catalyzed by cytochrome P450s.
  • FIG. 2A is a bar graph displaying the products of hexane catalysis of various BM-3 mutants.
  • FIG. 2B is a bar graph displaying the products of heptane catalysis of various BM-3 mutants.
  • FIG. 2C is a bar graph displaying the products of octane catalysis of various BM-3 mutants.
  • FIG. 2D is a bar graph displaying the products of nonane catalysis of various BM-3 mutants.
  • FIG. 2E is a bar graph displaying the products of decane catalysis of various BM-3 mutants.
  • FIG. 3A is a reaction schematic of the hydroxylation of dimethyl ether to produce formaldehyde.
  • FIG. 3B is a reaction schematic of Purpald with formaldehyde to form a purple colored adduct upon air oxidation (the first two compounds are colorless).
  • FIG. 4 is an illustration of A328 and A82 in the active site of wild type cytochrome P450 BM-3.
  • FIG. 5A is a GC/FID analysis of the (-)-menthyl carbonate diastereomers of the 2-octanol produced by 9-10A-A328V BM-3 catalyzed alkane oxidation.
  • FIG. 5B is a GC/FID analysis of the (-)-menthyl carbonate diastereomers of the 2-octanol produced by 1-12G BM-3 catalyzed alkane oxidation.
  • FIG. 6A is a GC/FID analysis of the octane hydroxylation product distributions using 9-10A-A328V as a purified protein.
  • FIG. 6B is a GC/FID analysis of the octane hydroxylation product distributions using 9-10A-A328V from a whole cell.
  • FIG. 7 is a depiction of a P450 molecule with the point mutations for the 1-12G mutant displayed as space filling structures.
  • Embodiments of the invention include mutant and altered forms of cytochrome P450 proteins.
  • mutants of cytochrome P450 BM-3 from Bacillus megaterium were engineered using an initial mutant P450 and a combination of directed evolution and site-directed mutagenesis, as discussed more completely below.
  • the starting mutant was a P450 enzyme with 11 mutations that allowed it to hydroxylate alkanes to produce certain amounts of particular enantiomeric and regiospecific alkane products.
  • the starting mutant was then engineered to display altered regio- and enantioselectivity towards various substrates ( e.g ., the new mutants have an altered hydroxylation profile).
  • the resulting enzymes were found to be capable of hydroxylating linear alkanes in an altered regio- and enantioselective manners.
  • the turnover number was high.
  • Each of the resulting P450 mutants produced regio- and enantiomeric products in different amounts. Simply put, the products of the initial P450 mutant were hydroxylated at particular positions in particular amounts, and the products of the new P450 mutants were hydroxylated at these particular positions, or in novel positions, in different amounts.
  • the proper mutant enzyme with these described characteristics, one can regio- and enantioselectively hydroxylate substrates in a desired manner. This provides tremendous benefits for specifically hydroxylating target substrates in a predefine manner.
  • mutant P450s with regio- and/or enantioselectivity Disclosed are mutant P450s with regio- and/or enantioselectivity.
  • one mutant P450, 9-10A-A328V was found to hydroxylate octane primarily at the 2-position to form S-2-octanol (40% ee).
  • Another mutant P450, 1-12G was found to hydroxylate alkanes larger than hexane primarily at the 2-position, but also formed R-2-alcohols (40-55% ee). These two mutants were discovered to have enhanced and altered regio- and enantiospecificity compared to other P450 enzymes, including the original 139-3 mutant from which they were derived.
  • mutant P450s that are capable of hydroxylating substrates as small as ethane.
  • one of the discovered mutants termed "1-12G” was advantageously found capable of hydroxylating ethane as a substrate.
  • regio-and enantioselective enzymes that are retained in whole-cell biotransformations with E . coli, where the engineered P450 enzymes are expressed at high levels, and the required cofactor is supplied endogenously.
  • mutants for the selective hydroxylation of alkanes to product well characterized products in known quantities.
  • all that is required to create a desired product is to select an appropriate mutant P450 enzyme that catalyzes a reaction to produce a desired enantio- or regiospecific product and then apply the substrate to the enzyme under conditions which allow for catalysis.
  • Methods of selecting and isolating the desired product from the products created are also known and disclosed herein.
  • mutant P450s that are capable of hydroxylating alkanes in a regio- and enantioselective manner.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity When percentage of sequence identity is used in reference to proteins or peptides it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art.
  • a "mutant" form of a protein or DNA molecule is a form that is altered from its wild-type composition. Mutant proteins typically have amino acid substitutions at one or more positions. Mutant DNA molecules typically have nucleotide substitutions in one or more positions. Mutant forms of a protein or DNA molecule can have the same, or altered, functions in comparison to the wild-type. For ease of discussion, mutants may be referred to by their variation from the single amino acid code from which the mutation arose.
  • the mutant in one format the mutant is referred to as XPOSY, where "X” refers to the single letter code of the amino acid in the original sequence, "POS” refers to the position of the mutation in the sequence, and Y refers to the single letter code for the new amino acid appearing at the mutation's position.
  • V175I would mean that in the original protein, the amino acid at position 175 is a valine (“V"), but in the mutant, the valine is replaced with an isoleucine ("I").
  • a "core mutation” is a mutation of a wild-type cytochrome P450 protein that provides the protein with enhanced alkane hydroxylase activity.
  • the cytochrome P450 protein is a P450 BM-3 protein. It should be realized that any mutation, or set of mutations, that enhance the ability of a cytochrome P450 protein to hydroxylate alkanes are considered core mutations.
  • a “core mutant” is a cytochrome P450 protein that has been altered to contain one or more core mutations.
  • a core mutant is the cytochrome P450 139-3 protein which was derived from mutations of P450 BM-3, and includes V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A295T, and L353V core mutations.
  • Those mutations that revert the amino acid sequence back to the wild type sequence for the selective hydroxylation mutations are not considered core mutations. Examples of which are H138, V178, and A295.
  • the core mutations will consist of V78A, T175I, A184V, H236Q, E252G, R255S, and L353V.
  • selective hydroxylation mutations or “selective mutations” are used interchangeably and refer to mutations that provide a P450 protein with altered regio- or enantio-selectivity towards substrates.
  • a protein having such mutations is termed a "selective hydroxylation mutant” or a “selective mutant”.
  • the target substrate of such mutants is an alkane. Examples of types or categories of selective hydroxylation mutants are discussed below, particularly in Tables 1-4.
  • the selective hydroxylation mutations may simply alter the selectivity of the P450 towards a single substrate, or across many substrates.
  • the selective mutation may alter both the selectivity and increase the functional ability of the enzyme, so that more regio or enantioselective end product is produced.
  • Non-limiting general examples of selective hydroxylation mutations include cytochrome P450 139-3 proteins having one or more of the following additional mutations: A328V, L75I, F87I and A82L.
  • Non-limiting examples of selective hydroxylation mutants showing altered or enhanced regioselective hydroxylation include cytochrome P450 139-3 proteins having one or more of the following additional mutations: A82I, and T260L. Examples of altered and enhanced regioselective and enantioselective mutants of cytochrome P450 139-3 can be found within Tables 1-4 and FIGs. 2A-2E .
  • Mutants may be both enantioselective and regioselective.
  • an enzyme is "regioselective" if the product that results from the enzymatic reaction is positioned in an altered position.
  • the enzyme is an alkane hydroxylase and the hydroxylation reaction results in a hydroxyl group positioned in an altered position. This means that while the original P450 may have created a first amount of product A and a second amount of product B, the regioselective enzyme could produce a third amount of product A and a fourth amount of product B.
  • the initial 139-3 mutant could be considered regioselective for particular substrates, the regioselective mutants described herein display different regioselectivity from the 139-3 mutant.
  • the product of a regioselective hydroxylase may contain a hydroxy group at the 2 position predominantly, rather than the 1 or the 3 position.
  • a distribution of hydroxyl groups in the final product that differs from the product of the wild-type enzyme can be sufficient to demonstrate that the enzyme is regioselective.
  • an increase of 1, 1-2, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-500 percent or more in the concentration of one product over another product is sufficient to demonstrate that the enzyme is regioselective.
  • an enzyme is regioselective when its selectivity is greater than the wild-type or 139-3 mutant P450 regioselectivity as shown in Table 2.
  • An enzyme is enantioselective if the hydroxylation reaction of the enzyme results in a high amount of one particular enantiomeric product compared to other possible enantiomeric products.
  • An enzyme that has an altered enantioselectivity means that while the original P450 may have created a first amount of enantiomeric product A and a second amount of enantiomeric product B, the enantioselective enzyme could produce a third amount of enantiomeric product A and a fourth amount of enantiomeric product B.
  • the initial 139-3 mutant could be considered enantioselective for particular substrates
  • the enantioselective mutants described herein display different enantioselectivity from the 139-3 mutant.
  • the enzyme is a mutant form of the wild-type P450 BM-3 enzyme.
  • An increase of 1, 1-2, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-500, or more in the concentration of one product over another product is sufficient to demonstrate that the enzyme is enantioselective.
  • the regioselective and enantioselective mutations are so characterized because of their activity to particular lengths of alkane substrate.
  • the regio- and enantioselective mutations display improved specificity compared to the 139-3 mutant, either in general or for a particular substrate.
  • the 139-3 mutation may be regioselective for octanes (61% produced as a 2-alcohol), it is not as regioselective as the 9-10A-A328V mutant (80%) or 1-12G mutant (82%) as described in more detail below.
  • the terms can be terms of degree.
  • any increase in selectivity either enantio-, regio, or both, might be sufficient as long as it is a measurable increase. For example, it may increase by 1, 1-5, 5-10, 10-20, 20-25, 25-30, 30-32, 32-40, 40-50, 50-100, 100-500 percent, or more, over the wild-type or over the 139-3 BM-3 mutant.
  • the regio- or enantioselectivity may only apply as an absolute; thus, an enzyme will only be regio- or enantioselective if the resulting mutant has a regio- or enantioselectivity where the 139-3 or wild-type BM-3 had none. This could happen in at least two ways.
  • Either the selectivity of the 139-3 mutant is effectively random, or the mutant BM-3 is active on a new substrate, and thus any selectivity would be more than the initial amount of selectivity of zero.
  • the level of activity is also important. Even though the wild type P450 may display a negligible amount of activity on alkanes, such activity, and any resulting regio- or enantioselectivity from the enzyme would not qualify the wild type P450 BM-3 as a regio- or enantioselective P450. This is because some effective, or substantial level of activity of the P450 on alkanes is still required. Such substantial or effective levels are discussed below; however, the wild type rate of catalysis in light of the total turnover is not substantial.
  • a “consistent" selective mutant is a selective mutant that displays a consistent bias of selectivity of product produced for more than one starting substrate.
  • the mutant 9-10A-A328V discussed below, is a consistent regioselective mutant for hexane, heptane, octane, nonane, and decane, as the products from hexane, heptane, octane, nonane, and decane all result predominantly in the 2-alcohol.
  • the 139-3 mutant results in more of the 3-alcohol for heptane substrates, but more of the 2-alcohol for the octane substrate.
  • a mutant P450 is a consistent regioselective enzyme if the largest amount of product produced from hexane, heptane and octane is the 2-alcohol. The majority of each of the products is made at the same position.
  • an improved enzyme or protein is "improved” if its activity is altered or enhanced from its parent composition.
  • an improved P450 139-3 protein is one that contains mutations and also exhibits regioselectivity, across substrates, or for an individual substrate, at a level that is above the regio- or enantioselectivity of the P450 139-3 protein.
  • the improved activity may be for a particular substrate, such as ethane, propane, hexane, heptane, octane, nonane, and/or, decane.
  • the improved regio- or enantioselectivity may provide the mutant with the ability to more effectively produce regio-selective products.
  • an increase of 1,1-5, 5-10,10-15,15-100,100-300, or more percent more effective than the wild-type or 139-3 mutant in converting the substrate to a single product is an improved regio- or enantiospecific enzyme.
  • Definitions and distinctions between the 139-3 mutant and the mutants described herein can be found in Tables 2, 3, and 4.
  • Another form of an "improved" mutant is one that effectively has a greater ability to efficiently produce a regio- or enantiospecific product.
  • the percentage of each product may not be very high, the efficiency of the formation of the products is great enough so that the desired product can be made in substantial amounts.
  • a wild type P450 BM-3 may have a product distribution of 17% 2-octanol, 40% 3-octanol, and 43% 4-octanol, it may have a relatively slow catalytic rate of 80 min -1 and less than about 100 total turnovers.
  • Improved mutants include those enzymes that have a higher catalytic rate, and/or higher turnover than the unimproved enzyme.
  • Predominant denotes the species of product that is the largest percent of the products made. Thus, given 4 products, three of which are equal, the fourth, if greater than the other three would be the predominant product.
  • a "hydroxylation profile" of a product is a description of the number and position of hydroxyl groups in the product.
  • an alkane hydroxylase enzyme typically creates products having a defined hydroxylation profile, such that hydroxyl groups are placed at certain positions on particular percentages of the final reaction products. Altering or modifying the hydroxylation profile of a product means changing the positions, or proportions, of hydroxyl groups in the final reaction products..
  • all of the products listed in Table 2 are used for the members of hydroxylation profile. For example, 1-alcohol, 2-alcohol, 3-alcohol, 4-alcohol and ketones may make up the hydroxylation profile.
  • Table 2 denotes the hydroxylation profiles of each of the mutants for substrates hexane through decane, in this embodiment.
  • a “variant” is distinguished from a mutant.
  • a variant P450 has at least one amino acid or nucleic acid difference from the wild-type P450.
  • a “variant” of a P450 mutant typically contains all of the mutant positions, plus additional changes in the amino or nucleic acid sequence.
  • the description "variant" will encompass sequences with changes, a variant of a P450 mutant will still maintain the amino acid or nucleotide changes that define the P450 mutant.
  • a protein that has one or more core mutations along with additional changes in its DNA or protein sequence is a "variant" core mutant.
  • variants can vary in the number and the types of residue replacements.
  • a variant may be any amino acid sequence that is 100-99, 99-98, 98-95, 95-90, 90-80, 80-70, 70-60, 60-50, or 50-30 percent identical to its original amino acid sequence.
  • a variant might be any amino acid sequence that is 100-99, 99-98, 98-95, 95-90, 90-80, 80-70, 70-60, 60-50, or 50-30 percent similar to the amino acid sequence of BM-3 P450.
  • a variant may be any amino acid sequence that is 100-99, 99-98, 98-95, 95-90, 90-80, 80-70, 70-60, 60-50, or 50-30 percent similar to the amino acid sequence of cytochrome P450 139-3.
  • the sequence of comparison may be a consensus sequence of known BM-3 proteins. These can apply, as appropriate, to both the amino acid sequence and nucleic acid sequences.
  • a variant may also be nucleic acid sequence that is capable of hybridizing to the disclosed BM-3 sequence under highly stringent or moderately stringent conditions. Highly stringent conditions are those that are at least as stringent as, for example, 4XSSC at 65° C., or 4XSSC and 50% formamide at 42° C.
  • the "active site" of the enzyme includes those residues which interact with the substrate in the binding and catalysis of the substrate. As appreciated by one of skill in the art, the precise residues involved in the active site may vary according to the substrate.
  • the active site may be defined through mutagenesis studies or through protein structures which will reveal which part of the enzyme is most closely interacting with the substrate.
  • the active site may be defined as those residues within a certain distance of the bound substrate or where the bound substrate would be positioned. For example, residues within 0-1, 1-2, 2-4, 4-5, 5-6, 6-7, 7-8, or 8-10 angstroms of the bound substrate, or the points at which the substrate will bind, are part of the active site.
  • the active site may include those residues within a certain distance of the heme group. For example, residues within 0-1, 1-2, 2-4, 4-5, 5-6, 6-7, 7-8, or 8-10 angstroms of the heme group, in the substrate bound or substrate free conformation, are part of the active site.
  • the active site may also be defined by the herein discussed crystal structures.
  • the active site may include the residues or mutants discussed herein which resulted in changes in activity of the P450 enzyme, consistent with a mutation in an active site.
  • the amino acids of the active site may include the amino acids at positions 75, 78, 82, 87, 88, 260, 328 of CYP102A1, or equivalent positions in variant proteins.
  • the area of the active site, but not all of the residues, is shown in FIG. 4 .
  • Amino acid A 328 is shown on the left, and A82 is displayed in the upper right. Palmitoyl glycine is displayed above the heme and between the two residues.
  • alkane is typically defined as a non-aromatic saturated hydrocarbon with the sequence of CnH(2n+2). For the purposes of this application and determining whether or not an enzyme is active with a particular substrate, an "alkane" does not encompass fatty acids that are the traditional targets for P450s.
  • the proteins disclosed include "conservative amino acid substitution variants" (i.e., conservative) of the proteins herein described.
  • a conservative variant refers to at least one alteration in the amino acid sequence that does not adversely affect the biological functions of the protein.
  • a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein.
  • the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
  • the amino acid sequence can often be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
  • proteins disclosed are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blastx, tblastn and tblastx Karlin et al., (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268 and Altschul, (1993) J. Mol. Evol. 36, 290-300 ) which are tailored for sequence similarity searching.
  • the approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.5 M sodium phosphate buffer at pH 7.2, 1 mM EDTA at pH 8.0 in 7% SDS at either 65° C. or 55° C., or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.05 M sodium phosphate buffer at pH 6.5 with 0.75 M NaCl, 0.075 M sodium citrate at 42° C.
  • a denaturing agent such as formamide, for example, 50% formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.05 M sodium phosphate buffer at pH 6.5 with 0.75 M NaCl, 0.075 M sodium citrate at 42° C.
  • Another example is use of 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate at pH 6.8, 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS and 10% dextran sulfate at 55° C., with washes at 55° C. in 0.2x.SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
  • nucleic acid molecule is said to be "isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
  • fragments of any one of the encoding nucleic acids molecules refers to a small portion of the entire protein coding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein.
  • fragments of the invention include fragments of DNA encoding mutant P450 BM-3 proteins that maintain altered or enhanced enantioselectivity and regioselectivity.
  • the encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a detectable label for diagnostic and probe purposes.
  • labels include, but are not limited to, fluorescent-labeled, biotin-labeled, radio-labeled nucleotides and the like.
  • a skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule.
  • the basic steps in directed evolution are (1) the generation of mutant libraries of polynucleotides from a parent or wild-type sequence; (2) (optionally) expression of the mutant polynucleotides to create a mutant polypeptide library; (3) screening/selecting the polynucleotide or polypeptide library for a desired property of a polynucleotide or polypeptide; and (4) selecting mutants which possess a higher level of the desired property; and (5) repeating steps (1) to (5) using the selected mutant(s) as parent(s) until one or more mutants displaying a sufficient level of the desired activity have been obtained.
  • the property can be, but is not limited to, alkane oxidation capability and enantio- and regiospecificity.
  • the parent protein or enzyme to be evolved can be a wild-type protein or enzyme, or a variant or mutant.
  • the parent polynucleotide can be retrieved from any suitable commercial or non-commercial source.
  • the parent polynucleotide can correspond to a full-length gene or a partial gene, and may be of various lengths.
  • Preferably the parent polynucleotide is from 50 to 50,000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the parent protein of interest may be used in the methods of this invention.
  • Any method for generating mutations in the parent polynucleotide sequence to provide a library of evolved polynucleotides including error-prone polymerase chain reaction, cassette mutagenesis (in which the specific region optimized is replaced with a synthetically mutagenized oligonucleotide), oligonucleotide-directed mutagenesis, parallel PCR (which uses a large number of different PCR reactions that occur in parallel in the same vessel, such that the product of one reaction primes the product of another reaction), random mutagenesis (e.g., by random fragmentation and reassembly of the fragments by mutual priming); site-specific mutations (introduced into long sequences by random fragmentation of the template followed by reassembly of the fragments in the presence of mutagenic oligonucleotides); parallel PCR (e.g., recombination on a pool of DNA sequences); sexual PCR; and chemical mutagenesis (e.g., by sodium bisulfollision
  • the evolved polynucleotide molecules can be cloned into a suitable vector selected by the skilled artisan according to methods well known in the art. If a mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector and/or express the mutant or variant protein or enzyme sequence. Any one of the well-known procedures for inserting expression vectors into a cell for expression of a given peptide or protein may be utilized. Suitable vectors include plasmids and viruses, particularly those known to be compatible with host cells that express oxidation enzymes or oxygenases. E . coli is one exemplary preferred host cell.
  • exemplary cells include other bacterial cells such as Bacillus and Pseudomonas, archaebacteria, yeast cells such as Saccharomyces cerevisiae, insect cells and filamentous fungi such as any species of Aspergillus cells.
  • plant, human, mammalian or other animal cells may be preferred.
  • Suitable host cells may be transformed, transfected or infected as appropriate by any suitable method including electroporation, CaCl.sub.2 mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, viral infection, or other established methods.
  • the mixed population of polynucleotides or proteins may then be tested or screened to identify the recombinant polynucleotide or protein having a higher level of the desired activity or property.
  • the mutation/screening steps can then be repeated until the selected mutant(s) display a sufficient level of the desired activity or property. Briefly, after the sufficient level has been achieved, each selected protein or enzyme can be readily isolated and purified from the expression system, or media, if secreted. It can then be subjected to assays designed to further test functional activity of the particular protein or enzyme: Such experiments for various proteins are well known in the art, and are described below and in the Examples below.
  • the evolved enzymes can be used in biocatalytic processes for, e.g., alkane hydroxylation:
  • the enzyme mutants can be used in biocatalytic processes for production of chemicals from hydrocarbons.
  • the enzyme mutants can be used in live cells or in dead cells, or it can be partially purified from the cells.
  • One preferred process would be to use the enzyme mutants in any of these forms (except live cells) in an organic solvent, in liquid or even gas phase, or for example in a super-critical fluid like CO 2 .
  • the organic solvent would dissolve high concentrations of the non-polar substrate, so that the enzyme could work efficiently on that substrate.
  • cofactor recycling methods well known in the art can be applied.
  • an enzyme capable of regenerating the cofactor, using a second substrate can be used.
  • the enzyme can be used in living cells, and the cofactor recycling can be accomplished by feeding the cells the appropriate substrate(s).
  • the NADPH and oxygen can also be replaced by a peroxide, for example hydrogen peroxide, butyl peroxide or cumene peroxide, or by another oxidant.
  • Mutations that enhance the efficiency of peroxide-based oxidation by BM-3 or other cytochrome P450 enzymes can serve to enhance the peroxide shunt activity of the enzyme mutants described here.
  • the mutations described here can be combined with such mutations, for example, and tested for their contributions to peroxide-driven alkane and alkene oxidation.
  • a screening method to detect oxidation comprises combining, in any order, substrate, oxygen donor, and test oxidation enzyme.
  • the assay components can be placed in or on any suitable medium, carrier or support, and are combined under predetermined conditions. The conditions are chosen to facilitate, suit, promote, investigate or test the oxidation of the substrate by the oxygen donor in the presence of the test enzyme, and may be modified during the assay.
  • the amount of oxidation product, i.e., oxidized substrate is thereafter detected using a suitable method.
  • a screening method can comprise a coupling enzyme such as horseradish peroxidase to enable or enhance the detection of successful oxidation.
  • one or more cofactors, coenzymes and additional or ancillary proteins may be used to promote or enhance activity of the test oxidation enzyme, coupling enzyme, or both.
  • test enzyme it is not necessary to recover test enzyme from host cells that express them, because the host cells are used in the screening method, in a so-called "whole cell” assay.
  • substrate, oxygen donor, and other components of the screening assay are supplied to the transformed host cells or to the growth media or support for the cells.
  • the test enzyme is expressed and retained inside the host cell, and the substrate, oxygen donor, and other components are added to the solution or plate containing the cells and cross the cell membrane and enter the cell.
  • the host cells can be lysed so that all intracellular components, including any recombinantly expressed intracellular enzyme mutant, can be in direct contact with any added substrate, oxygen donor, and other components.
  • the oxidation product may be a colored, luminescent, or fluorescent compound, so that transformed host cells that produce more active oxidation enzymes "light up” in the assay and can be readily identified, and can be distinguished or separated from cells which do not "light up” as much and which produce inactive enzymes, less active enzymes, or no enzymes.
  • a fluorescent reaction product can be achieved, for example, by using a coupling enzyme, such as laccase or horseradish peroxidase, which forms fluorescent polymers from the oxidation product.
  • a chemiluminescent agent, such as luminol can also be used to enhance the detectability of the luminescent reaction product, such as the fluorescent polymers.
  • Detectable reaction products also include color changes, such as colored materials that absorb measurable visible or UV light.
  • an alkane analog such as p-nitrophenoxy octane (8-pnpane), can be prepared that generates yellow color upon hydroxylation.
  • This "surrogate" substrate with a C8 backbone and a p-nitrophenyl moiety is an analog of octane, and allows use of a colorimetric assay to conveniently screen large numbers of P450 BM-3 or other cytochrome P450 mutants for increased hydroxylation activity in microtiter platens (Schwaneberg et al., 1999; Schwaneberg et al., 2001). Hydroxylation of 8-pnpane generates an unstable hemiacetal which dissociates to form (yellow) p-nitrophenolate and the corresponding aldehyde.
  • the hydroxylation kinetics of hundreds of mutants can then be monitored simultaneously in the wells of a microtiter plate using a plate reader (Schwaneberg et al., 2001). This method is particularly suitable for detecting P450 mutants with improved alkane-oxidation activity.
  • Enzyme mutants displaying improved levels of the desired activity or property in the screening assay(s) can then be expressed in higher amounts, retrieved, optionally purified, and further tested for the activity or property of interest.
  • the cytochrome P450 mutants created by directed evolution and selected for a desired property or activity can be further evaluated by any suitable test or tests known in the art to be useful to assess the property or activity.
  • the enzyme mutants can be evaluated for their alkane-oxidation capability, regio- and enantiomeric specificity.
  • An assay for alkane-oxidation capability essentially comprises contacting the cytochrome P450 mutant with a specific amount of alkane substrate, or a substrate which is an alkane analog such as 8-pnpane, in the presence of an oxygen donor, and any other components (e.g., NADPH) that are necessary or desirable to include in the reaction mixture, such as NADPH and buffering agents. After a sufficient incubation time, the amount of oxidation product formed, or, alternatively, the amount of intact non-oxidized substrate remaining, is estimated.
  • the amount of oxidation product and/or substrate could be evaluated chromatographically, e.g., by mass spectroscopy (MS) coupled to high-pressure liquid chromatography (HPLC) or gas chromatography (GC) columns, or spectrophotometrically, by measuring the absorbance of either compound at a suitable wavelength.
  • MS mass spectroscopy
  • HPLC high-pressure liquid chromatography
  • GC gas chromatography
  • V max maximum catalytic rate
  • Preferred substrates include, but are not limited to, methane, ethane, propane, butane, pentane, hexane, heptane, octane, and cyclohexane.
  • HPLC and GC techniques particularly when coupled to MS, can be used to determine not only the amount of oxidized product, but also the identity of the product.
  • octane can be oxidized to octanol where the hydroxyl group is positioned on any of the carbon atoms in the octanol molecule.
  • the substrates may also be used to determine regio- and enantiomeric specificity of the P450 enzymes.
  • Alkene-oxidation can be evaluated by methods similar to those described for alkanes, simply by replacing an alkane with the corresponding alkene, and designing an assay which promotes and detects epoxide formation of the alkene.
  • an assay which detects NADPH consumption may be used.
  • Preferred alkene substrates include ethene, propene, butene, pentene, hexene, heptene, and octene.
  • the 139-3 P450 BM-3 mutant exhibited significant activity on propane, despite the fact that small alkane substrates were not used to screen the mutant libraries in the directed evolution experiments. Because of this, as well as other factors, it was reasoned that decreasing the volume of the active site of the 139-3 mutant, using a combination of directed evolution and site-directed mutagenesis might further enhance this activity. Additionally, engineering the active site in this way might also confer regioselectivity towards longer alkanes — if the substrates are bound more tightly, fewer hydroxylation products may be possible. Of course this involves a delicate balance of decreasing the size of the active site as any alteration could result in making the active site so small as to prevent binding of the substrate. Additionally, the alterations could also prevent any activity by the enzyme as well.
  • the 11 mutations in the 139-3 P450 BM-3 are core mutations. All eleven of these mutations are preferably in the final P450 mutant in order for it to catalyze the oxidation of an alkane. As discussed further below, additional mutations give the P450 mutant the ability to hydroxylate the alkane enantio- and regioselectively.
  • a library of P450 BM-3 mutants was generated by performing a first round of directed evolution, wherein P450 BM-3 mutant 139-3 was combined with 15 other unsequenced P450 mutants of the same generation that also exhibited increased activity towards p-nitrophenyl octyl ether and octane.
  • the library was transformed into E . coli (DHS ⁇ ) competent cells, over-expressed, and lysed according to standard protocols developed by our laboratory. Aliquots of the cell-free extract of each mutant were transferred to 96-well plates where NADPH consumption was monitored in the presence of propane. Mutants identified from the screening process were then grown up and purified for comparative analysis using gas chromatography.
  • Mutant J was selected from the first round of directed evolution, based upon its increased rate of propane oxidation. This mutant was then used as the parent for the second round of evolution, the library for which was generated by error-prone PCR under conditions designed to yield 1 to 2 mutations in the heme domain of the P450 on average per gene.
  • Mutant 9-10A was selected from this library for its increased propane hydroxylation rate. The properties of these mutants are detailed in Tables 1-3 and FIG. 2 . Neither mutant J, nor mutant 9-10A acquired active-site mutations and showed no major changes in regioselectivity towards longer alkanes.
  • Mutant 9-10A was used to parent a third random-mutagenesis library.
  • a second screen was applied to this library to assess the amount of propane hydroxylation products generated by each mutant. This screen depended upon the surrogate substrate dimethyl ether, which is similar in size and C-H bond strength to propane.
  • dimethyl ether forms formaldehyde, which can be detected with Purpald dye ( Hopps, H.B. Aldrichim. Acta, 33, 28-30 (2000 )) ( FIG. 3A , showing the hydroxylation of the surrogate substrate dimethyl ether produces formaldehyde and FIG. 3B , showing that purpald reacts with formaldehyde to form a purple adduct upon air oxidation).
  • the third round of evolution did not produce a mutant with either increased propane hydroxylation activity or more propane hydroxylation products.
  • a possible explanation for this may be that further increases in activity require two or more simultaneous, or coupled, genetic mutations. Such events occur with very low probability and will not be found in screening a few thousand clones. Therefore, two residues were identified in the active site of mutant 9-10A as targets to modify by site-directed mutagenesis. The effect of these changes on alkane hydroxylation activity and product regioselectivity was then examined.
  • Crystal structures of wildtype P450 BM-3 with and without substrate reveal large conformational changes upon substrate binding at the active site ( Haines et al., Biochemistry, 40 (45):13456-13465 (2001 ); Li and Poulos, 1997; Paulsen and Ornstein, Proteins-Structure Function and Genetics, 21 (3):237-243 (1995 ); and Chang and Loew, (Biochemistry, 39 (10):2484-2498 (2000 )).
  • the substrate free structure displays an open access channel with 17 to 21 ordered water molecules. Substrate recognition serves as a conformational trigger to close the channel, which dehydrates the active site, increases the redox potential, and allows dioxygen to bind to the heme.
  • a tyrosine (Tyr51) at the entrance to the substrate-binding pocket makes a hydrogen bond to the carboxylate group of the substrate in the crystal structure of the enzyme bound with palmitoleic acid (Li and Poulos, 1997).
  • Arg 47 also at the entrance to the binding pocket, may form an ionic interaction as well.
  • Nonpolar alkane substrates must rely solely on hydrophobic partitioning into the enzyme's extended substrate channel, and poor substrate recognition may contribute to P450 BM-3's sluggish activity on octane and other alkanes or alkenes.
  • FIG. 4 shows the crystal structures of heme domain of wild type BM-3 containing a bound substrate.
  • Alanine 328 sits in the substrate binding pocket of BM-3 directly above the heme cofactor and is the closest residue in the protein to the proximal side of the heme iron. This residue and its mutation to valine in the wild type enzyme had been reported to affect substrate binding and turnover rates on fatty acids.
  • FIG. 4 shows the position of A328 and A82 in the active site of wild type cytochrome P450 BM-3. The illustration was made from the coordinates of the crystal structure 1JPZ.
  • the substrate is palmitoyl glycine and the terminal end ( ⁇ ) of the substrate is indicated.
  • Site-directed mutagenesis was used to change alanine 328 in 9-10A into the larger hydrophobic residue valine and determined the activity of this mutant (termed 9-10A-A328V) towards several alkanes. Neither the propane hydroxylation activity nor the total propane turnovers of this mutant improved relative to its parent, but a dramatic shift in its regioselective hydroxylation of longer alkanes was discovered. Wild type and all mutants of BM-3 generated by directed evolution were found to hydroxylate longer alkanes, such as heptane, octane, and nonane and form roughly equivalent distributions of 2-, 3-, and 4- alcohols.
  • Mutant 9-10A-A328V formed primarily (>80%) 2-alcohols with these substrates.
  • the resulting 2-alcohol was ⁇ 70% S -2-octanol (40% ee) (Tables 2, 3, FIG. 2 , in FIG. 2 , the first bar represents the 1-alcohol formed, the second bar represents the 2-alcohol formed and the third bar represents the 3-alcohol formed).
  • Other alkanes were not hydroxylated enantioselectively.
  • the second side chain in the BM-3 active site that was selected for alteration is located near the active site of the protein formed after the conformational change associated with substrate binding occurs.
  • the residue alanine 92 is located within 3.5 A of the terminal end of the substrate. ( Haines et al. Biochemistry, 40, 13456-13465 (2001 )). Given the proximity of this residue to the substrate, it is possible that changing this residue to a larger hydrophobic side chain could result in a decreased active site volume upon substrate binding.
  • the heme domain genes of J, 9-10A, 9-10A-A328V, and A82L 9-10A were recombined using DNA shuffling to generate a library and the library was then screened for improved propane activity using the NADPH consumption screen in the presence of propane and the dimethyl ether/Purpald screen.
  • the mutant with the highest activity, 1-12G, was selected from this library and its alkane hydroxylation activity was determined.
  • 1-12G contained all of the mutations introduced into the recombination library.
  • 1-12G is the double mutant A328V/A82L of 9-10A. Additionally, all of the background mutants present in the 139-9 original parent were also present in 1-12 G. Like the 9-10A-A328V, 1-12G hydroxylates alkanes at the 2-position (>80%). However, chiral GC analysis of these products revealed that 1-12G is enantioselective for the R -2-alcohols (40-55% ee), of heptane, octane, nonane, and decane (Tables 2, 3, FIG. 2 ).
  • the mutations that these mutants possess compared to the 139-3 mutant represent the regioselective hydroxylation mutations, as shown in Table 1. Additionally, as the 9-10A-A328V mutant is enantioselective over the 139-3 mutant, in creating the S-2 octanol, the mutations that are different between 9-10A-A328V and the 139-3 mutant represent the enantioselective mutants, and in particular the S-enantioselective mutants, as shown in Table 1.
  • the mutations that are different between 1-12G and the 139-3 mutant represent enantioselective mutants, and in particular the R-enantioselective mutants. These differences, as well as any others that are similarly discovered, represent the regio and enantioselective mutants.
  • amino acid positions (or corresponding positions in a different P450) that should be changed are illustrated in Table 1, so that one of ordinary skill in the art can produce a regioselective and/or enantioselective P450 enzyme.
  • the amino acids may be identical to those described in Table 1.
  • the residues may be conservative variants of those described in Table1. All of the residues that are characterized as regio- or enantioselective may be required in order to have a P450 protein that is regio- or enantioselective. May be only 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these residues are required.
  • the most important residues and guidance of which combination of residues or which additional residues can be changed for the same selectivity will be guided by the results presented herein, the tables showing the key residues, the crystal structure of P450, and the knowledge of one of ordinary skill in the art.
  • residues H138, V178, and A295 are core residues of P450 BM-3 as mutation of them results in a P450 enzyme with enhanced alkane hydroxylation activity.
  • these three residues are not mandatory for the altered or enhanced enantio- and regioselectivity of P450s, as these residues were not present on at least the 9-10A-A328V and 1-12G mutants.
  • mutant 1-12G is capable greater than the approximately 7000 turnovers reported here.
  • the addition of the A82L mutation to the 9-10A-A328V mutant overcame the enzyme's preference for octane, but in the process shifted the substrate in the binding pocket such that R -2-alcohols were the favored products ( FIG. 5 ).
  • This residue was selected for its proximity to the terminal end of the substrate in a crystal structure, but it is not clear if the larger leucine side chain "pushes" the substrate further up the active site or blocks the channel such that the substrate is flipped relative to its position in the A328V mutant.
  • the A82L mutation both with and without the A328V mutation conferred approximately an order of magnitude increase in stability as determined by total turnover number.
  • Lysates of E . Coli (DH5 ⁇ ) containing the overexpressed cytochrome P450 BM-3 mutants exhibit the same activity as purified protein, but still require the addition of expensive NADPH.
  • An isocitrate dehydrogenase-based NADPH regenerating system can be used to perform reactions using both cell lysate and purified protein with results indistinguishable from using NADPH alone.
  • the current embodiments could be used in an E . coli system as a whole cell catalyst since the alkane substrates and alcohol products should be permeable to the cell membrane.
  • whole cell cultures of DH5 ⁇ overexpressing the 9-10A-A328V and the 1-12G mutants were prepared.
  • alkanes shown above were perhaps the most difficult substrates to hydroxylate selectively as they have very little in the way of features which can be used to direct hydroxylation. As such, the active site mutants of 9-10A will be more selective on substrates with rigid shapes and functional groups that can only be bound in our active site in a single conformation.
  • the regio- and enantioselective enzymes will also allow the regio and enantioselective hydroxylation of nonfatty acids that are not considered alkanes.
  • the enzymes are able to specifically hydroxylate alkanes with other functional groups, alkenes, cyclic carbon groups of various sizes.
  • the mutant enzymes can regio- and enantioselectively hydroxylate any carbon which can be hydroxylated, and which is large enough to allow a limited number of binding positions in the binding site.
  • cyclized carbon groups being more rigid than alkanes, will allow regio- and enantioselective hydroxylation.
  • Variant 1-12G is active (approximately slightly less than 100 total turnovers) on ethane. This ethane activity appears to be the first reported by a cytochrome P450 enzyme. More broadly, it appears that the 1-12G variant is the first P450 capable of binding ethane. The particulars of how 1-12G interacts with the ethane substrate are discussed in more detail in the examples below.
  • additional mutations may be added to any of the above mutants in order to obtain enzymes with greater hydroxylation activity or altered or a higher degree of enantio- or regiospecificity.
  • additional mutations may be made in a selective manner to particular areas of the protein.
  • mutations at the active site that appear to further reduce or constrain a substrate should lead to an enzyme with the desired characteristics. Examples of such mutants and the resulting characteristics can be found in Table 4.
  • the background for the mutants on Table 4 was the 9-10A mutant. The total turnover rates for the mutants are comparable to that of 9-10A (or up to 50% better).
  • these mutations selected to minimize the space of the active site, resulted in varied regioselective enzymes, including enzymes that are capable of hydroxylating alkanes at the fourth position.
  • a favored position is defined as one in which at least 40% of the product exists.
  • One embodiment provides for a novel variant P450 BM-3 cytochrome P450 oxygenase in which one or more of the amino acid residues listed in Table 1A, which are not core residues, have been conserved.
  • the conserved residue is one that is different between either 9-10A-A82L and 139-3, 9-10-A328V and 139-3, and 1-12G and 139-3.
  • Conservation of an amino acid residue can show that the residue has an important function for the oxygenase activity and/or stability of the P450 enzyme.
  • the P450 BM-3 mutations identified herein to improve alkane-oxidation activity can simply be translated onto such non-P450 BM-3 enzymes to yield improved properties according to the invention.
  • sequence alignment software such as SIM (alignment of two protein sequences), LALIGN (finds multiple matching subsegments in two sequences), Dotlet (a Java applet for sequence comparisons using the dot matrix method); CLUSTALW (available via the World Wide Web as freeware), ALIGN (at Genestream (IGH)), DIALIGN (multiple sequence alignment based on segment-to-segment comparison, at University of Bielefeld, Germany), Match-Box (at University of Namur, Belgium), MSA (at Washington University), Multalin (at INRA or at PBIL), MUSCA (multiple sequence alignment using pattern discovery, at IBM), and AMAS (Analyse Multiply Aligned Sequences).
  • SIM alignment of two protein sequences
  • LALIGN finds multiple matching subsegments in two sequences
  • Dotlet a Java applet for sequence comparisons using the dot matrix method
  • CLUSTALW available via the World Wide Web as freeware
  • ALIGN at Genestream (IGH)
  • U.S. Pat. Publication No. 20030100744 has a representative sequence alignment (e.g ., in FIG. 20 and Table 2.)
  • the sequence alignments of P450 BM-3 with other cytochrome P450 enzymes can be taken from the literature, and amino acid residues corresponding to the mutated amino acid residues of the invention identified. For example, such information can be derived from de Montellano Cytochrome P450: Structure, Mechanism, and Biochemistry (Plenum Press, New York 1995 ), see, especially, FIG. I on page 187).
  • FIG. 7 An example of such a structure is demonstrated in FIG. 7 .
  • the P450 is displayed in a ribbon format, while the locations and shapes of the point mutations are displayed in space filling structures.
  • a functional variant is disclosed as a variant that functions as the mutant functions, but has different mutations that allow it to so function. Such variants are described herein, as the process for identifying such variants has been fully described in the making of the mutant itself.
  • This example demonstrates one method by which recombination of the P450 BM-3 mutants may occur, as done in the first 1st library recombination.
  • the first generation of mutants was created by StEP recombination of mutant 139-3 (V78A, H138Y, T175I, V178I, A194V, H236Q, E252G, R255S, A290V, A295T) with 15 other mutants from the same generation. ( Glieder et al., Nature Biotech., 20, 1135-1139 (2002 )), Zhao et al., Nature Biotechnology, 16, 258-261 (1998 )).
  • a mutant, J, (V78A, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V) was isolated based on its increased NADPH depletion rate using propane as a substrate.
  • Table 2 displays the enantio- and regiospecific qualities of the J mutant. For example, the J mutant produces less of the 2-alcohol than the 139-3 mutant does. However, the J mutant creates more of the 3-alcohol than the 139-3 mutant does.
  • Table 3 displays the rate of NADPH depletion as 3000 min -1 for octane and 600 min -1 for propane. These values are clearly higher than the 2000 and 100 rates for the 139-3 mutant P450.
  • the distribution of the products can be seen in FIG. 2 and in Table 2.
  • This distribution of products is also known as the hydroxylation profile.
  • the profile for, for example, octane if 1%, 52%, 25%, 22%, and 5%, for the 1-alcohol, 2-alcohol, 3-alcohol, 4-alcohol, and ketone respectively.
  • the hydroxylation profile is also 57% (S) ee.
  • the J mutant clearly has an altered hydroxylation profile. For example, for hexane, the J mutant produces 23% of the product in the 2-alcohol form, while the 139-3 mutant only produces 14% of hexanes in the 2-alcohol form.
  • This example demonstrates one method by which random mutagenesis of P450 BM-3 may be achieved, as described in the 2nd and 3rd library steps above.
  • the second and the third generation were created by error-prone PCR using the Genemorph kit (Strategene, La Jolla, CA) according to the manufacturer's protocol, using approximately 50 ng (x ng for third) of plasmid DNA as template and primers BamHI-forw (5'-ggaaacaggatccatcgatgc-3'; SEQ ID NO: 55 ) and SacI-rev (5'-gtgaaggaataccgccaagc-3'; SEQ ID NO: 56 ).
  • Mutant 9-10A (R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, AS90V, L353V) was isolated from the 2 nd generation based on increased NADPH consumption and NADP+ formation using propane as a substrate. No increase in activity was observed in the products of the third library.
  • Table 3 displays the catalytic properties of the 9-10A mutant produced from this step. While the rate of octane synthesis has not changed, relative to the J mutant, the rate of propane synthesis has actually decreased, although both are still substantially above the rates for the 139-3 mutant. Of course, the total turnover for 9-10A, 1100, is greater than the total turnover for J, 800. The distribution of the products can be seen in FIG. 2 and in Table 2.
  • This example demonstrates one method by which site directed mutagenesis may be performed, as described above in the 3 rd and 4 th generations.
  • Base substitution mutations were introduced into mutant 9-10A by PCR overlap extension mutagenesis.
  • Position A82 was mutated to L, I, V and F using mutagenic primers A82forw (5'-ggagacgggttatttacaagc-3'; SEQ ID NO: 57 ) and A82rev (6'-gcttgtaaataacccgtctccaanaaaatcacg-3'; SEQ ID NO: 58 ).
  • Position A328 was mutated to V using mutagenic primers A328V forw (5'-gcttatggccaactgttcctgc-3'; SEQ ID NO: 59 ) and A328V rev (5'-gcaggaacagttggccataagc - 3'; SEQ ID NO: 60 ).
  • A328V forw 5'-gcttatggccaactgttcctgc-3'; SEQ ID NO: 59
  • A328V rev 5'-gcaggaacagttggccataagc - 3'; SEQ ID NO: 60 .
  • two separate PCRs were performed, each using a perfectly complementary primer (BamHI-forw and SacI-rev) at the end of the sequence and a mutagenic primer.
  • the resulting two overlapping fragments that contain the base substitution were then annealed together in a second PCR to amplify the complete mutated gene.
  • Mutant 9-10A-A82L was isolated based on increased turnover of dimethyl ether.
  • the properties of the resulting mutants, 9-10A-A82L and 9-10A-A328V can be observed in Tables 2 and 3.
  • 9-10A-A82L demonstrated a decrease in catalytic rate for octane and propane, while it displayed a significant increase in the total turnover rate for octane and propane. It also showed a regioselectivity that favors the 4-alcohol product, at least for the octane, nonane, and decane products, in contrast to the 139-3 mutant.
  • the 9-10A-A328V mutant showed both a decrease in rate and turnover, as compared to the 9-10A mutant.
  • this mutant displayed a high degree of regioselectivity, as shown in Table 2 and FIG. 2 (identified as A328V), especially for the 2-alcohol.
  • the distribution of the products for the hydroxylation profiles can be seen in FIG. 2 and in Table 2.
  • the 9-10A-A328V mutant produced 76% of its product in that form.
  • the hydroxylation profile of this mutant is different from the 139-3 mutant.
  • Mutant 1-12G has all of the mutations from the 9-10A-A328V mutant, with an additional mutation at position A82L. Interestingly, the rate of catalysis of this mutant is only 400 min -1 and 20 min -1 for octane and propane. However, the total turnover, as shown in Table 3 (7500 for octane and 6020 for propane), is much higher than for any of the other mutants. Additionally, as can be observed in FIG. 2 , the bias towards the production of 2-alcohol products is much greater than for any of the other mutants as well, as high as 86% for decane. As discussed below, this mutant is also capable of hydroxylating ethane, something the wild type and 139-3 mutant are effectively unable to do.
  • the data in Table 2 demonstrate the altered hydroxylation profile of the 1-12G mutant.
  • the hydroxylation profile of the 139-3 mutant for octane was 1, 61, 20,17, and 5 percent for the 1-, 2-, 3-, 4-alcohols and ketone respectively
  • the same values for the 1-12G mutant were 5, 82, 11, 3, and 1.
  • the enzyme produces a larger percent of the 2-alcohol than it did before.
  • the hydroxylation profile of the enantiomeric products for octane has changed significantly between 139-3 and 1-12G, as the 139-3 results in 58(S), while 1-12G results in 39(R).
  • P450 BM-3 was expressed and purified as described previously. ( Glieder et al., Nature Biotech., 20, 1135-1139 (2002 )).
  • E. coli DH5 ⁇ transformed with these plasmids, was used for expression of P450 BM-3 on a 500 mL scale as well as for expression in 96-well plates.
  • Enzyme concentration was measured in triplicated by CO-difference spectra. ( Omura et al., Journal of Biological Chemistry, 239, 2370-2378 (1964 )). The characteristics of the enzymes were then tested, as described in the following examples.
  • This example demonstrates one method by which cell lysates can be prepared for high-throughput screening.
  • Single colonies were picked and inoculated by a Qpix robot (Genetix, Beaverton, OR) into 1-mL deep-well plates containing Luria-Bertani (LB) medium (350 ⁇ L, supplemented with 100 mg/mL ampicillin). The plates were incubated at 30°C, 250 rpm, and 80% relative humidity. After 24 hours, clones from this preculture were inoculated using a 96-pin replicator into 2-mL deep-well plates containing TB medium (400 ⁇ L, supplemented with 100mg/mL ampicillin, 10 ⁇ M IPTG and 0.5 mM ALA).
  • the first mutant library was screened for NADPH depletion using propane as substrate. 170 ⁇ L of phosphate buffer (0.1 M, pH 8.0), saturated with propane was added to 30 uL of E. coli supernatant. The reaction was initiated by addition of 50 uL NADPH (0.8 mM and NAPDH oxidation was monitored at 340 nm for five min using a Spectramax Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA).
  • the second library was additionally screened for NADP + formation using propane as substrate as described earlier. ( Glieder et al., Nature Biotech., 20:1135-1139 (2002 ); Tsotsou et al. Biosens. Bioelectron., 17, 119-131 (2002 )).
  • residual NADPH was destroyed with acid after an appropriate amount of time followed by conversion of NADP + to a highly fluorescent alkali product at high pH which was then measured fluorometrically.
  • the results of the screens were used to select the J mutant and 9-10A mutant discussed above.
  • This example demonstrates direct methods for the high-throughput determination of enzymatic activity.
  • a screen based on the demethylation of dimethyl ether was used in the later generations.
  • 120 ⁇ L of phosphate buffer (0.1 M, pH-8) saturated with dimethyl ether was added.
  • NADPH 50 ⁇ L, 1.0 mM
  • Purpald 168 mM in 2 M NaOH
  • the purple color was read approximately 15 min later at 500 nm using a Spectramax Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA). The results demonstrated that the third round of mutagenesis did not produce a mutant with improved results.
  • This example demonstrates one method by which one can determine the enzyme kinetics of an enzyme.
  • the enzymes were purified and quantified as described above. Initial rates of NADPH consumption were measured at 25°C in a BioSpec-1601 UV/VIS spectrophotometer (Shimadzu, Columbia, MD). For the liquid alkanes, substrate stock solutions in ethanol (10 ⁇ L) were added to the protein solution (100 nM, final concentration) and incubated for 2 min before initiating the reaction by addition of 200 ⁇ L NADPH (0.8 mM) and the absorption at 340 nm was monitored. Rates for any given substrate concentration were determined in triplicate. Results are shown in Table 3. The 9-10A mutant has the highest rate, while the 1-12G mutant has the lowest rate.
  • This example demonstrates one method by which one can determine if the enzyme is capable of alkane hydroxylation reactions.
  • Reactions with the liquid alkanes hexane, heptane, octane, nonane, and decane were performed in closed 20 mL scintillation vials and stirred at low speed using magnetic stirring bars.
  • purified protein or cell lyse
  • the substrates were added to this solution as 50 ⁇ L of 400mM ethanol solutions to make 4 mM total substrate and 1% ethanol.
  • the magnesium sulfate was removed by filtration, and 1 ⁇ L of pyridine and 2.5 ⁇ L (-)-menthyl chloroformate was added: After one hour, 1 mL of deionized water was added to the reaction. After vortexing and letting the layers separate, the organic phase was removed with a pipet and dried with anhydrous magnesium sulfate. The drying agent was again removed with a pipet filter and the remaining solution analyzed by gas chromatography. Control reactions were performed by repeating these steps without the addition of substrate and revealed no background levels of these specific products. Results are summarized in Table 2 and in FIGs. 2A-2E.
  • FIG. 5 shows a graph from a GC analysis of the (-)-menthyl carbonate diasteroemers of the 2-octanol produced by mutant BM-3 catalyzed alkane oxidation.
  • S-2-octanol elutes at 18.4 (18.393 and 18.410) minutes
  • R-2-octanol elutes at 18.6 (18.553 and 18.575) minutes.
  • This example demonstrates one method by which propane hydroxylation may be performed and monitored.
  • Propane hydroxylation reactions were performed in 25 mL Schlenk flasks and no co-solvent was used in the reaction.
  • enzyme either purified or in cell lysate
  • 500 all of NADPH-regeneration system containing 1 mM NADP + , 100 mM sodium isocitrate, and 20 Units/mL isocitrate dehydrogenase was quickly added.
  • the flask was topped with a balloon filled with equal amounts of propane and dioxygen.
  • the cells were collected by centrifugation at 3500 rpm for 10 minutes and resuspended in 20 mL of 0.2M potassium phosphate buffer pH-7.4 containing 0.5% glucose, 100 ⁇ g/mL ampicillin, 1 mM IPTG, and 0.5 mM ⁇ -aminolevulonic acid, 5 mM alkane (from a 500 mM stock of alkane in dimethyl sulfoxide). This mixture was shaken for 8 hours at 37°C and 250 rpm. Product distributions were measured by gas chromatography after extracting this culture with 1 mL of chloroform.
  • This example demonstrates how gas chromatography can be used for identification and quantification of analytes. Identification and quantification of analytes were performed using purchased standards and 5 point calibration curves with internal standards. All analyses were injected at a volume of 1.0 ⁇ L and performed at least in triplicate. Analysis of hydroxylation products were performed on a Hewlett Packard 5890 Series II Plus gas chromatograph with both a flame ionization (FID) and electron capture detector (ECD) and fitted with a HP-7673 autosampler system.
  • FID flame ionization
  • ECD electron capture detector
  • the (-)-menthyl chloroformate derivatized chiral products were separated as diastereomers on a CycloSil-B chiral capillary column (Agilent Technologies, 30 m length, 0.32 mm ID, 0.25 ⁇ m film thickness) connected to the FID detector.
  • Each pair of diastereomers required a different temperature program to fully resolve the pair, but a typical program is as follows: chiral heptanol analysis - 250°C injector, 300°C detector, 100°C oven for 1 minute, then 10°C/minute gradient to 180°C, hold at 180°C for 10 minutes, 10°C/minute gradient to 250°C, then 250°C for 3 minutes.
  • the propyl nitrite products were analyzed with an HP-1 capillary column (crosslinked 1% phenyl methyl siloxane, 30 m length, 0.32 mm ID, 0.25 ⁇ m film thickness) connected to an ECD detector.
  • the temperature program for separating 1- and 2-propyl nitrites was 250°C injector, 300°C detector, 30°C oven for 3 minutes, 20°C/minute gradient to 200°C, 200°C for 5 minutes. Results can be observed in FIG. 2 , FIG. 5 , FIG. 6 , and Tables 2 and 3.
  • the replaced alcohol component in the ester can be a chromophore, such as p-nitrophenolate, or can be reacted with a dye to form a chromophore, such as vinyl alcohol. This will then allow the presence of the chiral alcohol product to be detected colorimetrically in the presence of the lipase and the ester substrate, as discussed in greater detail in Konarzycka-Bessier et al., Angewandte Chemie International English Edition, 42(12): 1418-1420 (2003 ). Alternatively, alcohol dehydrogenases that selectively oxidize chiral alcohols can be incorporated into a coupled screening system.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Claims (17)

  1. Isolierte P450 Enzymmutante umfassend eine Sequenz wie in SEQ ID NR: 2 aufgeführt und mit den Mutationen R47C, V78A, A82L, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V und L353V, worin die P450 Enzymmutante Alkane hydroxyliert.
  2. Isolierte P450 Mutante nach Anspruch 1, worin das Enzym eine Mutante des Cytochrom P450 BM-3 Enzyms ist.
  3. Isolierte P450 Mutante nach Anspruch 1, weiter umfassend die Mutation A328V.
  4. Isolierte P450 Mutante nach einem der vorstehenden Ansprüche, worin die P450 Mutante eine selektive Hydroxylierung aufweist.
  5. Isolierte P450 Mutante nach einem der vorstehenden Ansprüche, worin die P450 Mutante eine selektive Aktivität umfasst, die beständig bei Hexanen, Dekanen, Heptanen, Oktanen und Nonanen wirkt.
  6. Isolierte P450 Mutante nach einem der vorstehenden Ansprüche, worin die P450 Mutante eine Alkanhydroxylierungsaktivität umfasst, die zu dem gleichen regiospezifischen Produkt für Hexan, Heptan, Oktan, Nonan und Dekan für mindestens 40 % des durch die Alkanhydroxylierungsaktivität hergestellten Gesamtprodukts führt.
  7. Isolierte P450 Mutante nach einem der vorstehenden Ansprüche, worin die P450 Mutante einen Gesamtumsatz für Oktan von mehr als 1000 aufweist.
  8. Isolierte P450 Mutante nach einem der vorstehenden Ansprüche, worin die P450 Mutante eine maximale Katalyserate von 2001 (min-1) aufweist.
  9. Mutiertes BM-3 P450 Enzym nach Anspruch 1,
    worin die P450 Enzymmutante eine veränderte Enantiospezifität aufweist verglichen mit der Enantiospezifität der Mutante 139-3 umfassend SEQ ID NR: 2 mit den Mutationen V78A, H138Y, T175I, V178I, H236Q, E252G, R255S, A295T und L353V, oder
    worin die P450 Enzymmutante eine veränderte Enantiospezifität aufweist verglichen mit der Regiospezifität der Mutante 139-3.
  10. Isolierte P450 Enzymmutante nach Anspruch 1, weiter umfassend eine Alkanhydroxylierungsaktivität gegen Ethan.
  11. Verfahren zur Herstellung einer P450 Enzymmutante mit veränderten selektiven Hydroxylierungseigenschaften, wobei das Verfahren umfasst:
    Bereitstellen einer ersten P450 Mutante, die die Fähigkeit besitzt, Alkane eines Substrats zu Hydroxylieren, um ein Produkt mit einem ersten Hydroxylierungsprofil herzustellen,
    Verändern von mindestens einer Aminosäure in der ersten P450 Mutante, um eine zweite P450 Mutante herzustellen, wobei die zweite P450 Mutante die Fähigkeit besitzt, Alkane des Substrats zu Hydroxylieren, um ein Produkt mit einem zweiten Hydroxylierungsprofil herzustellen, wobei die zweite Mutante die folgenden Mutationen aufweist: R47C, V78A, A82L, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V und L353V.
  12. Verfahren nach Anspruch 11,
    wobei die Veränderung der mindestens einen Aminosäure durch gerichtete Evolution erreicht wird, oder
    wobei die Veränderung der mindestens einen Aminosäure durch ausgewählte Punktmutationen erreicht wird, vorzugsweise wobei die Punktmutationen in einer aktiven Stelle der P450 Mutante ausgewählt werden.
  13. Isolierte Nukleinsäure, die eine Cytochrom P450 Mutante nach einem der Ansprüche 1-10 kodiert.
  14. Verfahren zum Hydroxylieren eines Alkans in selektiver Art und Weise, wobei das Verfahren umfasst:
    Bereitstellen einer isolierten Mutante eines P450 Enzyms nach einem der Ansprüche 1-10, und
    In Kontaktbringen der isolierten P450 Mutante mit dem Alkan,
    wobei die isolierte P450 Mutante das Alkan in selektiver Art und Weise hydroxyliert.
  15. Verfahren nach Anspruch 14,
    wobei die selektive Art und Weise eine regioselektive Art und Weise ist, oder
    wobei die selektive Art und Weise eine enantioselektive Art und Weise ist.
  16. Verfahren nach Anspruch 14, wobei die isolierte P450 Mutante das Alkan mit einer Umsatzrate von mehr als 100 hydroxyliert.
  17. Verfahren nach Anspruch 14, wobei das Alkan
    ein Oktan ist und die isolierte P450 Mutante das Oktan mit einer Umsatzrate von mehr als 1000 hydroxyliert, oder
    ausgewählt ist aus der Gruppe bestehend aus Dekan, Nonan, Oktan, Heptan, Hexan, Pentan, Propan und Ethan.
EP04776528A 2003-06-17 2004-06-15 Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450 Expired - Lifetime EP1639091B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US47912603P 2003-06-17 2003-06-17
PCT/US2004/018832 WO2005017105A2 (en) 2003-06-17 2004-06-15 Regio- and enantioselective alkane hydroxylation with modified cytochrome p450

Publications (3)

Publication Number Publication Date
EP1639091A2 EP1639091A2 (de) 2006-03-29
EP1639091A4 EP1639091A4 (de) 2008-09-17
EP1639091B1 true EP1639091B1 (de) 2012-12-05

Family

ID=34193029

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04776528A Expired - Lifetime EP1639091B1 (de) 2003-06-17 2004-06-15 Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450

Country Status (3)

Country Link
US (5) US7524664B2 (de)
EP (1) EP1639091B1 (de)
WO (1) WO2005017105A2 (de)

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7226768B2 (en) * 2001-07-20 2007-06-05 The California Institute Of Technology Cytochrome P450 oxygenases
EP1639091B1 (de) 2003-06-17 2012-12-05 California University Of Technology Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450
WO2006105082A2 (en) * 2005-03-28 2006-10-05 The California Institute Of Technology Alkane oxidation by modified hydroxylases
US8715988B2 (en) * 2005-03-28 2014-05-06 California Institute Of Technology Alkane oxidation by modified hydroxylases
US8252559B2 (en) * 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
EP2069513A4 (de) * 2006-08-04 2012-03-21 California Inst Of Techn Verfahren und systeme zur selektiven fluorinierung organischer moleküle
US9828597B2 (en) 2006-11-22 2017-11-28 Toyota Motor Engineering & Manufacturing North America, Inc. Biofunctional materials
US8101399B2 (en) * 2006-12-12 2012-01-24 The Regents Of The University Of California Artemisinic epoxide and methods for producing same
US20080248545A1 (en) * 2007-02-02 2008-10-09 The California Institute Of Technology Methods for Generating Novel Stabilized Proteins
US20080268517A1 (en) * 2007-02-08 2008-10-30 The California Institute Of Technology Stable, functional chimeric cytochrome p450 holoenzymes
US8802401B2 (en) * 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates
GB0719620D0 (en) 2007-10-08 2007-11-14 Isis Innovation Mutant Enzymes
DE102008054918A1 (de) * 2008-12-18 2010-07-01 Evonik Degussa Gmbh Verfahren zur enzymatischen Umsetzung von Alkanen
KR101190881B1 (ko) 2009-08-24 2012-10-12 전남대학교산학협력단 박테리아 사이토크롬 피450을 이용한 심바스타틴 또는 로바스타틴의 사람에서의 대사산물의 신규한 생산방법 및 이를 위한 조성물
WO2011053478A1 (en) * 2009-10-26 2011-05-05 Saudi Arabian Oil Company Visible light-enhanced enzymatic promotion of hydrocarbon reactions
US8361769B1 (en) 2009-11-16 2013-01-29 U.S. Department Of Energy Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme
US8309333B1 (en) 2009-11-16 2012-11-13 The United States of America, as represented by Department of Energy Regioselective alkane hydroxylation with a mutant AlkB enzyme
CA2794387C (en) 2010-03-28 2019-02-19 Novozymes A/S Enzymatic hydroxylation of aliphatic hydrocarbon
US11015149B2 (en) 2010-06-21 2021-05-25 Toyota Motor Corporation Methods of facilitating removal of a fingerprint
US10988714B2 (en) 2010-06-21 2021-04-27 Regents Of The University Of Minnesota Methods of facilitating removal of a fingerprint from a substrate or a coating
US9388370B2 (en) 2010-06-21 2016-07-12 Toyota Motor Engineering & Manufacturing North America, Inc. Thermolysin-like protease for cleaning insect body stains
US8796009B2 (en) 2010-06-21 2014-08-05 Toyota Motor Engineering & Manufacturing North America, Inc. Clearcoat containing thermolysin-like protease from Bacillus stearothermophilus for cleaning of insect body stains
US9121016B2 (en) 2011-09-09 2015-09-01 Toyota Motor Engineering & Manufacturing North America, Inc. Coatings containing polymer modified enzyme for stable self-cleaning of organic stains
EP2426198A1 (de) 2010-09-03 2012-03-07 B.R.A.I.N. Biotechnology Research And Information Network AG Cytochrome P450 Monooxygenase Varianten
US9322007B2 (en) 2011-07-22 2016-04-26 The California Institute Of Technology Stable fungal Cel6 enzyme variants
EP2906518A4 (de) 2012-10-09 2016-07-27 California Inst Of Techn Durch hämenzyme katalysierte in-vivo- und in-vitro-olefincyclopropanierung
WO2014058729A1 (en) * 2012-10-09 2014-04-17 California Institute Of Technology In vivo and in vitro carbene insertion and nitrene transfer reactions catalyzed by heme enzymes
EP3450549B1 (de) 2012-10-29 2020-09-30 University Of Rochester Artemisininderivate, verfahren zu deren herstellung und deren verwendung als antimalariamittel
RU2016116253A (ru) 2013-09-27 2017-11-01 Кодексис, Инк. Автоматизированный скрининг вариантов фермента
US9399762B2 (en) 2014-02-18 2016-07-26 California Institute Of Technology Methods and systems for sulfimidation or sulfoximidation of organic molecules
WO2015176020A1 (en) 2014-05-16 2015-11-19 Provivi, Inc. Synthesis of olefinic alcohols via enzymatic terminal hydroxylation
US9708587B2 (en) 2014-07-09 2017-07-18 Codexis, Inc. P450-BM3 variants with improved activity
WO2017007547A1 (en) 2015-07-07 2017-01-12 Codexis, Inc. Novel p450-bm3 variants with improved activity
EP3858986A1 (de) * 2020-02-03 2021-08-04 Bayer Aktiengesellschaft P450-bm3-monooxygenase-varianten zur c19-hydroxylierung von steroiden
WO2021168228A1 (en) * 2020-02-21 2021-08-26 Dow Silicones Corporation Method of preparing silanols with selective cytochrome p450 variants and related compounds and compositions
CN113388589B (zh) * 2021-05-28 2022-11-22 湖北大学 一种细胞色素p450单加氧酶cyp109b2突变体及其应用

Family Cites Families (66)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4599342A (en) * 1984-01-16 1986-07-08 The Procter & Gamble Company Pharmaceutical products providing enhanced analgesia
US5785989A (en) * 1985-05-01 1998-07-28 University Utah Research Foundation Compositions and methods of manufacturing of oral dissolvable medicaments
GB8723662D0 (en) 1987-10-08 1987-11-11 British Bio Technology Synthetic gene
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5198346A (en) * 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5429939A (en) * 1989-04-21 1995-07-04 Kirin Beer Kabushiki Kaisha DNA sequences useful for the synthesis of carotenoids
FR2658300B1 (fr) * 1990-02-13 1992-05-29 Abx Sa Reactif et methode d'utilisation de celui-ci pour la numeration automatique des leucocytes basophiles du sang en appareils de mesure par variation de resistivite.
US5340971A (en) * 1990-09-17 1994-08-23 Metrologic Instruments, Inc. Automatic bar code reading system having selectable long range and short range modes of operation
JPH0767394B2 (ja) 1991-03-20 1995-07-26 三洋化成工業株式会社 化学発光増強剤、化学発光利用検出・定量法及びキット
US6335160B1 (en) 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US6117679A (en) 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5837458A (en) * 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
US5565483A (en) * 1995-06-07 1996-10-15 Bristol-Myers Squibb Company 3-substituted oxindole derivatives as potassium channel modulators
CN1212015A (zh) 1995-11-01 1999-03-24 Bg公开有限公司 突变单加氧酶细胞色素P450cam
US5965408A (en) * 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US5789166A (en) * 1995-12-08 1998-08-04 Stratagene Circular site-directed mutagenesis
US5741691A (en) * 1996-01-23 1998-04-21 California Institute Of Technology Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media
EP0910645B1 (de) * 1996-03-19 2004-04-28 Millennium Pharmaceuticals, Inc. KINASE FÜR DIE STELLEN-SPEZIFISCHE PHOSPHORYLIERUNG VON I-kappa-B-alpha
US6096548A (en) 1996-03-25 2000-08-01 Maxygen, Inc. Method for directing evolution of a virus
JP3376838B2 (ja) * 1996-11-05 2003-02-10 トヨタ自動車株式会社 プレニル二リン酸合成酵素
US6326204B1 (en) 1997-01-17 2001-12-04 Maxygen, Inc. Evolution of whole cells and organisms by recursive sequence recombination
EP2261373A3 (de) 1997-01-17 2011-12-14 Codexis Mayflower Holdings, LLC Evolution prokaryotischer ganzer Zellen durch rekursive Sequenzrekombination
IL131966A0 (en) 1997-03-18 2001-03-19 Novo Industri As An in vitro method for construction of a dna library
US6153410A (en) 1997-03-25 2000-11-28 California Institute Of Technology Recombination of polynucleotide sequences using random or defined primers
US6361988B1 (en) * 1997-03-25 2002-03-26 California Institute Of Technology ECB deacylase mutants
KR20010032861A (ko) * 1997-12-08 2001-04-25 캘리포니아 인스티튜트 오브 테크놀로지 폴리뉴클레오티드 및 폴리펩티드 서열을 형성하는 방법
US5945325A (en) * 1998-04-20 1999-08-31 California Institute Of Technology Thermally stable para-nitrobenzyl esterases
US20030077795A1 (en) * 1999-03-10 2003-04-24 Wilson C. Ron Cytochrome P450 monooxygenase and NADPH Cytochrome P450 oxidoreductase genes and proteins related to the omega hydroxylase complex of candida tropicals and methods relating thereto
US6902918B1 (en) 1998-05-21 2005-06-07 California Institute Of Technology Oxygenase enzymes and screening method
ATE547532T1 (de) 1998-06-29 2012-03-15 Bristol Myers Squibb Co Verfahren zur erzeugung von hochgradig diversen bibliotheken
KR20010083086A (ko) 1998-07-28 2001-08-31 추후제출 기능적 진핵세포 단백질의 발현
EP1102861A1 (de) * 1998-08-05 2001-05-30 University Of Pittsburgh Modellierung organischer verbindung reaktivität in vermittelten reaktionen des cytochrom p450
FR2782323B1 (fr) 1998-08-12 2002-01-11 Proteus Procede de production in vitro de sequences polynucleotidiques recombinees, banques de sequences et sequences ainsi obtenues
EP1117777A2 (de) 1998-09-29 2001-07-25 Maxygen, Inc. Shuffling von gene mit veränderten kodons
GB9825421D0 (en) 1998-11-19 1999-01-13 Isis Innovation Process for oxidising terpenes
WO2000042560A2 (en) 1999-01-19 2000-07-20 Maxygen, Inc. Methods for making character strings, polynucleotides and polypeptides
US6090604A (en) * 1999-02-24 2000-07-18 Novo Nordisk Biotech, Inc. Polypeptides having galactose oxidase activity and nucleic acids encoding same
US6524837B1 (en) * 1999-03-29 2003-02-25 California Institute Of Technology Hydantoinase variants with improved properties and their use for the production of amino acids
GB9914373D0 (en) * 1999-06-18 1999-08-18 Isis Innovation Process for oxidising aromatic compounds
ATE371028T1 (de) * 1999-07-27 2007-09-15 Basf Ag Modifizierte cytochrom p450-monooxygenasen
US6787343B2 (en) * 1999-11-12 2004-09-07 Washington State University Research Foundation Cytochrome P450 oxygenases and their uses
US20010051855A1 (en) * 2000-02-17 2001-12-13 California Institute Of Technology Computationally targeted evolutionary design
AU2001238397A1 (en) 2000-02-17 2001-08-27 California Institute Of Technology Computationally targeted evolutionary design
US6498026B2 (en) * 2000-02-25 2002-12-24 Hercules Incorporated Variant galactose oxidase, nucleic acid encoding same, and methods of using same
US7115403B1 (en) * 2000-05-16 2006-10-03 The California Institute Of Technology Directed evolution of galactose oxidase enzymes
US20020045175A1 (en) * 2000-05-23 2002-04-18 Zhen-Gang Wang Gene recombination and hybrid protein development
US7465567B2 (en) * 2001-04-16 2008-12-16 California Institute Of Technology Peroxide-driven cytochrome P450 oxygenase variants
US7226768B2 (en) * 2001-07-20 2007-06-05 The California Institute Of Technology Cytochrome P450 oxygenases
WO2003008562A2 (en) 2001-07-20 2003-01-30 Creighton University Methods for detecting genes associated with bone properties, height, and/or body mass properties
WO2003091835A2 (en) 2001-10-26 2003-11-06 California Institute Of Technology Computationally targeted evolutionary design
AU2003256264A1 (en) 2002-06-04 2003-12-19 California Institute Of Technology Carotenoid synthesis
EP1639091B1 (de) 2003-06-17 2012-12-05 California University Of Technology Regio- und enantioselektive Alkanhydroxylierung mit modifiziertem Cytochrom p450
US8603949B2 (en) * 2003-06-17 2013-12-10 California Institute Of Technology Libraries of optimized cytochrome P450 enzymes and the optimized P450 enzymes
US6906930B2 (en) * 2003-06-26 2005-06-14 Delta Electronics, Inc. Structure and method for an isolated boost converter
US7435570B2 (en) * 2003-08-11 2008-10-14 California Institute Of Technology Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use
WO2006105082A2 (en) 2005-03-28 2006-10-05 The California Institute Of Technology Alkane oxidation by modified hydroxylases
US8715988B2 (en) * 2005-03-28 2014-05-06 California Institute Of Technology Alkane oxidation by modified hydroxylases
US8252559B2 (en) * 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
EP2069513A4 (de) * 2006-08-04 2012-03-21 California Inst Of Techn Verfahren und systeme zur selektiven fluorinierung organischer moleküle
WO2008085900A2 (en) 2007-01-05 2008-07-17 The California Institute Of Technology Methods for generating novel stabilized proteins
US20080248545A1 (en) * 2007-02-02 2008-10-09 The California Institute Of Technology Methods for Generating Novel Stabilized Proteins
US20080268517A1 (en) * 2007-02-08 2008-10-30 The California Institute Of Technology Stable, functional chimeric cytochrome p450 holoenzymes
WO2008118545A2 (en) 2007-02-08 2008-10-02 The California Institute Of Technology Methods for generating novel stabilized proteins
WO2008115844A2 (en) 2007-03-16 2008-09-25 The California Institute Of Technology Stable, functional chimeric cytochrome p450 holoenzymes
US8802401B2 (en) * 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates

Also Published As

Publication number Publication date
EP1639091A4 (de) 2008-09-17
US20090298148A1 (en) 2009-12-03
WO2005017105A2 (en) 2005-02-24
US20150010976A1 (en) 2015-01-08
US8741616B2 (en) 2014-06-03
EP1639091A2 (de) 2006-03-29
US7863030B2 (en) 2011-01-04
US7524664B2 (en) 2009-04-28
US9145549B2 (en) 2015-09-29
US8343744B2 (en) 2013-01-01
WO2005017105A3 (en) 2007-11-15
US20050059128A1 (en) 2005-03-17
US20110244537A1 (en) 2011-10-06
US20130273626A1 (en) 2013-10-17

Similar Documents

Publication Publication Date Title
US9145549B2 (en) Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US9322001B2 (en) Cytochrome P450 oxygenases
Grogan Cytochromes P450: exploiting diversity and enabling application as biocatalysts
Peters et al. Regio-and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3
US7704715B2 (en) Peroxide-driven cytochrome P450 oxygenase variants
US20090264311A1 (en) Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use
JP2010517579A (ja) 修飾ヒドロキシラーゼによるアルカン酸化
CA2332615A1 (en) Dna shuffling of monooxygenase genes for production of industrial chemicals
WO2006051729A1 (ja) P450遺伝子の単離法
Buergler et al. Process intensification for cytochrome P450 BM3‐catalyzed oxy‐functionalization of dodecanoic acid
Makino et al. Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol
JP4969448B2 (ja) シトクロムｐ４５０の生物学的変換反応系

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20051222

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL HR LT LV MK

DAX Request for extension of the european patent (deleted)
RIN1 Information on inventor provided before grant (corrected)

Inventor name: MEINHOLD, PETER

Inventor name: PETERS, MATHEW W.,GEVO INCL.

Inventor name: ARNOLD, FRANCES, H.

PUAK Availability of information related to the publication of the international search report

Free format text: ORIGINAL CODE: 0009015

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 1/20 20060101ALI20080123BHEP

Ipc: C07H 21/04 20060101ALI20080123BHEP

Ipc: A61K 38/44 20060101ALI20080123BHEP

Ipc: C12Q 1/34 20060101ALI20080123BHEP

Ipc: C12N 9/00 20060101AFI20080123BHEP

Ipc: C07K 16/00 20060101ALI20080123BHEP

Ipc: C12Q 1/68 20060101ALI20080123BHEP

Ipc: C12P 21/04 20060101ALI20080123BHEP

Ipc: C12Q 1/00 20060101ALI20080123BHEP

Ipc: C12P 21/06 20060101ALI20080123BHEP

Ipc: C12N 15/00 20060101ALI20080123BHEP

Ipc: C07K 1/00 20060101ALI20080123BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20080814

RIN1 Information on inventor provided before grant (corrected)

Inventor name: MEINHOLD, PETER

Inventor name: PETERS, MATHEW W.,GEVO INCL.

Inventor name: ARNOLD, FRANCES, H.

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602004040315

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: C12N0001000000

Ipc: C12N0009020000

RIC1 Information provided on ipc code assigned before grant

Ipc: C07K 14/32 20060101ALI20120507BHEP

Ipc: C12N 9/02 20060101AFI20120507BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 587307

Country of ref document: AT

Kind code of ref document: T

Effective date: 20121215

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602004040315

Country of ref document: DE

Effective date: 20130131

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 587307

Country of ref document: AT

Kind code of ref document: T

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130316

REG Reference to a national code

Ref country code: NL

Ref legal event code: VDEP

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130306

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130305

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20130405

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

26N No opposition filed

Effective date: 20130906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602004040315

Country of ref document: DE

Effective date: 20130906

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

Effective date: 20140228

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130615

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130630

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130701

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20121205

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20040615

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20130615

REG Reference to a national code

Ref country code: DE

Ref legal event code: R082

Ref document number: 602004040315

Country of ref document: DE

Representative=s name: 2K PATENT- UND RECHTSANWAELTE PARTNERSCHAFT MB, DE

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20230626

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230627

Year of fee payment: 20

REG Reference to a national code

Ref country code: DE

Ref legal event code: R071

Ref document number: 602004040315

Country of ref document: DE